Abstract Theentericnervoussystem(ENS)accomplishesabroadrangeofactivitiesthatrelyonaremarkably diversepopulationofneuronalandglialsubtypes.Lossofspecificcelltypes,suchasnitricoxide(NO) producing neurons (nitrergic neurons) leads to enteric neuropathies associated with dysmotility disordersincludingesophagealachalasia,gastroparesisandinfantilehypertrophicpyloricstenosis.The underlying pathophysiology of these disorders have remained largely unknown due to limitations of currently available cellular models. We have recently reported a new alternative approach for differentiation of ENS lineages from human pluripotent stem cells under fully defined conditions, providing a unique and reliable framework for ENS disease modeling and drug discovery. Taking advantageofhighcontentchemicalcompoundscreeningincombinationwithfatemapreconstruction guided by single cell transcriptomics, here we propose a new strategy for efficient derivation and prospective isolation of enteric nitrergic neurons. This system will provide a unique in vitro model for identificationofpharmacologicalregulatoroftheseneuronsanddissectionofcellularmechanismsthat underlieGIdysmotilty.Wewillfurtherevaluatethepotentialoftheseneuronsintransplantationstudies aimedattheultimatedevelopmentofcell-basedtreatmentofentericneuropathiesrelatedtotheloss ofnitrergicneurons.